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Nano Today (2010) 5, 213—230

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REVIEW

Functionalisation of nanoparticles for biomedicalapplications

Nguyen T.K. Thanha,b,∗, Luke A.W. Greena,b

a The Davy Faraday Research Laboratories, The Royal Institution of Great Britain, 21 Albemarle Street, London W1S 4BS,United Kingdomb Department of Physics and Astronomy, University College London, Gower Street, London WC1E 6BT,United Kingdom

Received 26 March 2010; received in revised form 4 May 2010; accepted 6 May 2010

KEYWORDSNoble metal;Nanoparticles;Quantum dot;Magnetism;Fluorescence;Biofunctionalisation;Biomedicine;Synthesis

Summary Nanoparticles with cores composed of inorganic materials such as noble, magneticmetals, their alloys and oxides, and semiconductors have been most studied and have vastpotential for application in many different areas of biomedicine, from diagnostics to treatmentof diseases. The effects of nanoparticles must be predictable and controllable, and deliver thedesired result with minimum cytotoxicity. These criteria can be met by careful tailoring of theligand shell, allowing stabilisation, specific targeting and recognition of biochemical species.For these reasons, this review is focused on the synthesis and biofunctionalisation of inorganicmetal, semiconductor and magnetic nanoparticles for biomedical applications.© 2010 Elsevier Ltd. All rights reserved.

Introduction

Nanoparticles (NPs) are attracting considerable interest asviable biomedical materials and research into them is grow-ing due to their unique physical and chemical properties.These NPs can be composed of a variety of materials includ-ing noble metals (e.g. Au [2], Ag [3,4], Pt [5], Pd [6]),semiconductors (e.g. CdSe, CdS, ZnS [2,7], TiO2 [8], PbS[9], InP [9], Si [10]), magnetic compounds (e.g. Fe3O4

[11], Co [12], CoFe2O4 [13], FePt [6], CoPt [14]) and their

∗ Corresponding author at: The Davy Faraday Research Laborato-ries, The Royal Institution of Great Britain, 21 Albemarle Street,London W1S 4BS, United Kingdom. Tel.: +44 2074916509.

E-mail address: [email protected] (N.T.K. Thanh).

combinations (core—shell NPs and other composite nanos-tructures). Biomedical applications of NPs include drugcarriers, labelling and tracking agents [2,7], vectors for genetherapy, hyperthermia treatments and magnetic resonanceimaging (MRI) contrast agents [5,15]. In order for the NPsto be useful in biomedicine, they must satisfy certain crite-ria. For in vitro applications such as fluorescent staining ofproteins and TEM imaging, NPs must outperform the conven-tional agents while having minimal cytotoxicity. In vivo, NPshave to avoid non-specific interactions with plasma proteins(opsonisation) and either evade or allow uptake by the retic-uloendothelial system (RES) depending on the application,to reach their intended target efficiently. They must alsomaintain colloidal stability under physiological conditions,preferably including a wide range of pH. NPs carrying a pay-load, such as drug molecules or DNA for gene therapy must

1748-0132/$ — see front matter © 2010 Elsevier Ltd. All rights reserved.doi:10.1016/j.nantod.2010.05.003


214 N.T.K. Thanh, L.A.W. Green

avoid premature release, yet specifically deliver the load tothe desired site. Chemical modification of the NP surfaceis necessary for specific interactions with biomolecules ofinterest.

Synthesis of nanoparticles

Synthesis in water

Nano-structured materials including Au [16,17], Ag [4], Co[3], Nix [18] Fe3O4, Fe2O3, �-FeO(OH) [19], SiO2 [20] andCdTe [21,22] have been synthesised in aqueous solution.These methods provide water-dispersible NPs, a necessityfor the application in biological systems; however, controlover particle size distribution is still limited for many semi-conductor and magnetic NP systems. NP size affects theproperties so a narrow size distribution is essential [23]. Thiscan be reduced post-synthesis by using a hydrophobic ligandsuch as oleic acid to transfer the NPs to an apolar solvent,from which particles of the desired size may be obtained bysize-selective precipitation. However, this is a tedious, lowyielding multistep procedure and the particles are then nolonger in the desired medium, water [24].

Gold nanoparticles can be prepared by reduction of Au(III) salts using reducing agents such as citric acid, sodiumcitrate, sodium ascorbate or amines [25]. Iron (hydr)oxidesare synthesised by the alkali coprecipitation process, withthe composition and morphology of the resultant NPsdepending on the precise reaction conditions [19,26]. Alkaliprecipitation is also suited for the preparation of more com-plex multi-metallic ferrites [27]. Borohydride reduction isanother approach suited for the preparation of NPs com-posed of Co, Fe, Au and CdTe [3,25,28—30]. Ag NPs canbe synthesised following a green synthesis protocol wherestarch was used as a stabiliser [4]. Synthesis of SiO2 is wellestablished via the Stober method [20]. Success has alsobeen achieved in the synthesis of water soluble Co andCoPt structures through the use of multithiol ligands, ther-moresponsive polymers and thioether end-functionalisedpolymers [3,31,32].

Synthesis of nanoparticles in non-polar solvents

Syntheses in organic solvents have been published for a widerange of NPs composed of noble metals, transition metals,oxides and semiconducting materials [3,33—36]. The growthof NPs, the crystal structure and the cessation of the growthdepend on the environment and are fundamentally regu-lated by the ligands. Thus, in the absence of ligand, particlesurfaces are exposed, so they fused together and precipi-tate. These ligands tend to be either surfactant species suchas fatty acids or alkane thiols, rendering the particles highlyhydrophobic. In some cases the solvent itself acts as a lig-and, semiconductor quantum dots (QDs) were capped withtri-n-octyl phosphine oxide (TOPO) [36].

Water soluble NPs may be prepared in a one-step syn-thesis in organic solvents by judicious choice of stabilisationagents. Amphiphilic ligands such as peptides [12] and ther-moresponsive polymers [31], have been used to produce CoNPs.

Synthesis of nanoparticles by other methods

Other methods for NP synthesis have been reported includ-ing gel templating and solvent-free methods such aschemical vapour deposition (CVD) [37], electrical explosion[38] and mechanical milling [39].

In gel templating, metal salts are reduced in a porousgel matrix. The choice of gel is crucial to the mecha-nism; agarose gel has an inherent reducing effect [40].Closely related to gel templating is the synthesis of sub-nanometre Au NPs in the dendrimer polyamidoamine. Again,this method leads to very small Au NPs, termed ‘‘gold quan-tum dots’’ due to the fluorescence exhibited by both thedendrimer [41] and the Au core in this size regime [41—43].

CVD, mechanical milling and other solvent-free methodsare especially useful for the synthesis of other interest-ing materials such as NdFeB and carbon nanotubes forwhich there are no established ‘‘wet’’ synthesis routes[44]. To date, NPs prepared by CVD have been used almostexclusively for heterogeneous catalysis, magnetic data stor-age and nanoelectronic devices rather than biomedicine.

Figure 1 Synthetic pathways to biomedically applicable nanoparticles, the dotted line indicates that materials synthesised insolid state are not water soluble or may have no biomedical application.


Functionalisation of nanoparticles 215

Electrical explosion and mechanical milling are currentlyunsuited to production of NPs for biomedicine due to theirrelatively large particle size and poor control over size dis-tribution (Fig. 1).

Water solubilisation of nanoparticles

Water solubilisation may be carried out either as the finalstage of the functionalisation process of NPs, or as anintermediate stage. It should be noted that the terms‘‘solubilisation’’ and ‘‘solution’’ when applied to NPs doesnot refer to the solvation of the inorganic cores but ratherthe physically and chemically stable colloidal suspensionswhere NPs do not aggregate, dissociate, or chemically reactto the solvent or any dissolved gas with time. Water solu-bilisation refers to the conjugation of colloidally unstableNPs with hydrophilic ligands to give stable NPs in aqueoussolution.

Ionic stabilisation

Functional groups on the surface of NPs allow conjugationwith ionic ligands. With the molecule bound to the nanopar-ticle, the charge resides on the outside of the particle givingway to coulombic repulsion and thus dispersion of the NPs.

Once the charge screening is large enough, the extent ofthe coulombic repulsion can be diminished by the addition ofa salt, leading to precipitation of NPs or ‘‘salting out’’ [45].As the physiological salt concentration is around 100 mM [46]this is generally sufficient to cause precipitation of NPs lack-ing additional stabilisation. For this reason, unfortunately,ionically stabilised NPs are generally unsuitable for biomedi-cal application. The stability of NPs coated with species suchas citric acid/citrate [16], orthophosphoric acid/phosphate[47] and other species that may easily gain or lose protons ishighly sensitive to pH as protonation/deprotonation affectsthe surface charge (�-potential). If the magnitude of the�-potential is reduced below the point at which coulom-bic repulsion is effective, NPs will aggregate. This occursat pH values around the pKa of the surface functional group,making acidic anionic ligands (phosphate, citrate) suitablefor stabilisation in basic to mild acidic conditions [48,49],whereas cationic ligands such as alkylammoniums offer sta-bilisation from acidic to mildly alkaline conditions [33].

While ionic stabilisation alone is generally insufficient toprevent aggregation of NPs, ionic interactions with chargedspecies in biological media can have a significant effect onthe overall stability of the ligand shell and the NPs’ func-tion. Positively charged NPs tend to be removed rapidly fromthe blood, ending up predominantly in the liver and spleenwhereas negative NPs have a longer circulation time and aremainly taken up by the lymph nodes [50]. Charge neutralitymay be achieved by using either uncharged or zwitterionicligands with no overall charge. Neutral ligands must be bulkyin order to compensate for the lack of coulombic repul-sion, leading to a larger hydrodynamic radius and a generallylonger circulation time in the blood. Zwitterionic ligands, onthe other hand, have been reported to yield NPs with smallerhydrodynamic radii and much lower degrees of opsonisation[51]. Coulombic repulsion is also useful as it can protectligands on the NPs from exchanging with biomolecules [52];

the disadvantage of highly charged NPs is that they are morereadily opsonised [53].

However, inhomogeneous surface chemistry, segregatedsurface charge distribution and detachment of ligands in var-ious environments can compromise the reproducibility andlong-term stability. Therefore, the stability of the systemshould be tested for a wide range of electrolyte concentra-tions, values of pH at various time points so that its validitycan be verified.

Steric stabilisation

An alternative to ionic stabilisation is to provide a physicalbarrier to prevent aggregation. Steric stabilisation can beachieved by coating NPs with a ligand shell or embeddingthem with an inorganic or polymeric matrix.

Polymeric ligandsPolymers make excellent ligands as they surround the NPswith a substantial physical barrier preventing the core NPsfrom coming into contact. The consequence of this enhancedcore separation is an increase in the hydrodynamic radiusof the NPs [54]. This is desirable for in vivo applicationsrequiring a long circulation time, but disadvantageous ifrapid diffusion to the extravascular space is required; essen-tially, size is a very important factor in the biodistributionof NPs [51]. There are many suitable polymeric ligands forthe provision of water solubility (Table 1 ), the most com-mon of which are based on poly(ethylene glycol) (PEG) andcarbohydrates such as starch [55], dextran [56] and chitosan[57].

PEG is especially suitable as a ligand for NPs requiringlong circulation times in blood, as it reduces the degree ofopsonisation [51] and provides excellent long-term stabilityin high salt concentrations and pH extremes [58—60]. Conju-gation and alteration of the head groups of PEG derivativesnot only allows selective attachment to NP surfaces, but alsomakes way for biofunctionalisation [60].

Like small-molecule stabilising agents [61], the concen-tration of polymeric stabilisers may be used to controlthe NP core morphology [3,62]. As a result of the poly-merisation method and reaction conditions used, variousNP structures are formed. Such structures include sin-gle microparticles incorporating multiple NP cores [63,64],individual core—shell systems [65] and cases where the poly-mer is larger than the NP, leading to a templating effect[42,66].

Small-molecule ligandsThe advantage of small molecules as ligands is that theyoffer a certain degree of physical barrier, similar to poly-meric ligands, but give a smaller hydrodynamic radius. Invivo applications require a small hydrodynamic radius forefficient trans membrane permeation and excretion [51].However, care must be taken not to make the molecularshell too thin, as this leads to an insufficient steric barrier,resulting in reduced NP stability [82] and aggregation [83].Molecular species suitable for water solubilisation of NPstend to incorporate functional groups allowing ionic stabili-sation and further (bio)chemical modification once in water.A common small-molecule form of PEG is an �-thiol func-



Table 1 Different polymers used to coat NPs.

Polymer Ligand addition method Core Chemical functional groups

PAMAMa [42,67] Ligand addition in methanol Au Amine, amide groups.Dendrimer provides a stericframework.

PGAMAb and PLAMAc [68] Reduction of chloroauricacid in the presence ofglycopolymer

Au Multiple —OH groups offerstability, solubility andresistance to high saltconcentrations.

PEG-phosphine Ligand addition Au Multidentate —PO groupsoffer binding to Au, thehydrophobic tails offersolubility in organicsolvents.

PMAA-DDTd [3,62] Reduction of CoCl2 andchloroauric acid in thepresence of polymer

Co, Au —COOH group offersstability, solubility andmorphology control.

PVPe [69] Ligand exchange viahydrophobic interactions

CdSe/ZnS QDs, Au, Fe2O3 Amide within pyrollidonestructure gives watersoluble properties. PVP actsas a stabilising agent forcoupling to aminofunctionalised colloids.

PAA-octylamine [70] Carbodiimide coupling inthe presence of QDs

CdSe, CdS, ZnS QDs —OH surface coatingminimises non-specificcellular binding.

PAA modifiedf [71] Carbodiimide coupling tocreate the multidentateligand, followed by ligandexchange

CdTe QDs —SH, NH2 groups interactwith QDs to give a smallhydrodynamic radius and nodegradation of opticalproperties.

PMAOg-block-PEG [72] Ligand addition byhydrophobic interactions

CdSe QDs —COOH groups offerbiofunctionalisationpossibilities, ester groupson hydrophilic side chainsoffer water solubility.

PBAh-block-PEAi-block-PMAA [73] Ligand addition byhydrophobic interactions

CdSe, —ZnS QDs Alkyl chains givehydrophobic interactions,while the —COOH groupsgive hydrophilicity andfurther functionalisation.

Pyr-PDMAEMAj [74] Ligand exchange with TOPO CdSe QDs Pyrene is a fluorescenttracer

PMPC-PGMAk [75] Coprecipitation of ferricand ferrous salts in thepresence of the blockpolymer

Fe3O4 PO4− and NMe4

+ mimicphospholipid head groups ofcell membranes, while theglycerol group holds a1,2-diol group whichcreates a five memberedchelate between glycerolresidue and the NP.

ptBAl [1] Ligand exchange followedby CuI catalysed ‘clickchemistry’ andpolymerisation

Fe2O3 PO43− and COO−.

PNIPAMm [63,76] Silica coating andfunctionalisation followedby precipitationpolymerisation

Fe3O4, —CONH— allows hydrogenbond networking.



Table 1 (Continued)

Polymer Ligand addition method Core Chemical functional groups

PLLAn and PCLo [77] In situ polymerisation withSnII initiator

Fe3O4 —COOC— and chiral natureare biocompatible.

PMEMAp [65] Surface activation and atomtransfer radicalpolymerisation

Fe3O4 —COC— and —COOC—.

PVAq [78,79] Alkali coprecipitation offerric and ferrous chloridesfollowed by direct ligandaddition

�-Fe2O3 and Fe3O4 —OH groups make hydrogelsand a physical barrier.

PAAr [80] Coprecipitation in thepresence of polymer

�-Fe2O3 and Fe3O4 —COOH gives pH adjustable,solubility and stability inwater at pH >5.

PNIPco-t-Bams [31,81] Thermal decomposition inthe presence of polymer

Fe2O3 and Co Amphiphilic, allowing singlestep syntheses, —CONH—groups offer hydrogen bondnetworking.

a Poly(amidoamine).b Poly(D-glucosamido ethylmethacrylate).c Poly(2-lactobionamido ethylmethacrylate).d Alkyl thioether end-functionalised poly(methacrylic acid).e Poly(vinyl pyrollidone).f PAA modified with cysteamine and ethylene diamine.g Poly(maleic anhydride-alt-1-octadecene).h Poly(butylacrylate).i Poly(ethylacrylate).j Pyrene-poly(dimethylaminoethyl methacrylate).k Poly[2-(methacryloyloxy)ethyl phosphorylcholine]-block-(glycerol monomethacrylate).l �-Acetylene-poly(tert-butyl acrylate).

m Poly(N-isopropylacrylamide).n Poly(L,L-lactic acid).o Poly(�-caprolactone).p Poly(2-methoxyethyl methacrylate).q Poly(vinyl alcohol).r Poly(acrylic acid).s Poly(N-isopropyl-co-t-butylacrylamide).

tionalised alkane ether of tetra(ethylene glycol) which iscommonly used for water solubilising and stabilisation of AuNPs [84]. The exposed end of the ethylene glycol chain canalso be modified to provide chemical functionality or ionicstabilisation [52,85].

Phase transfer (PT)

Almost all biochemical reactions are conducted in an aque-ous environment. However, due to the synthetic methodsgenerally used, NPs are capped with hydrophobic ligands,meaning they are unstable in aqueous suspension. In orderto overcome this barrier, a variety of PT methods have beendeveloped to transfer NPs from organic to aqueous solu-tion. PT agents include tetraalkylammonium salts such astetraoctylammonium bromide (TOAB) for transfer of Au NPs;4-(dimethylamino)pyridine (DMAP) for transfer of Au andPd NPs [34], hexadecyltrimethylammonium bromide (CTAB)for magnetic NPs (MNPs), [58,86] and other amphiphilicspecies such as 2,3-dimercaptosuccinic acid (DMSA) [87], �-cyclodextrin [88] and copolymers [89] for transfer of oleicacid capped NPs. These PT agents act as labile ligands, being

easily replaced with the desired biofunctional ligand aftertransfer is complete.

An alternative to using labile PT agents is to carry out aligand exchange/ligand addition step in the organic phase,using an amphiphilic ligand capable of forming a strongNP-ligand bond (Fig. 2). A common example of this is mer-captoundecanoic acid and other mono- and dimercaptoalkane carboxylic acids [42,83,90]. The advantage of thissystem is that the carboxylic acid terminal group providesnot only water solubility but also a site for further chemicalfunctionalisation.

A different approach takes a partially solvent-freeligand exchange and PT process in which phosphine oxide-terminated PEG is mixed with OA-capped NPs in THF to giveNPs which can then be dispersed in water [91].

Ligand exchange

Ligand exchange with increased binding strength is commonfor the formation of self-assembled monolayers of alkanethiols and their derivatives on the surface of Au NPs coatedwith ionic stabilisation agents such as citrate or tetraocty-



Figure 2 Ascorbic acid binding to iron in an octahedral man-ner.

lammonium bromide (TOAB). The sulfur-gold bond is one ofthe most frequently used bonds in the functionalisation ofNPs, having a bond strength of approximately 210 kJ mol−1

[92].The alternative to exchanging weakly bound ligands for

strongly binding ligands is to replace a strongly bound lig-and with a ligand present in high concentration during theexchange procedure. This drives the equilibrium towards thethermodynamically less stable NPs coated with weakly bind-ing ligands; thus satisfying Le Chatellier’s principle (Eq. (1)),where n and m are the number of molecules of X and Yrespectively.

K = [NP—Xn][Y]m

[NP—Xm][X]n (1)

This approach can be used to replace strongly bound oleicacid with ethanol [1] and to introduce peptide sequencesto PEGylated QDs [93]. However, due to the relatively highprevalence of potential ligands in vivo, including thiols, car-boxylic acids, peptides, sugars and phosphates; ligands onNPs intended for in vivo use must have a high enough affin-ity for the NP that they will not undergo significant exchangewith these species [94].

Oxygen based ligandsNeutral ligands. This class of ligands includes alcohols,polyols, polyethers, carbonyls and carbohydrates. In gen-eral, there is little coordination chemistry between NPs andsimple alcohols as they are insufficiently electron-donatingto serve as good ligands. Moreover, the size-selective pre-cipitation process relies on addition of ethanol, methanol,acetone and other polar solvents, to alter the polarity suchthat NPs are no longer stable in solution, precipitatingat different polarities dependent on their size. However,serotonin has been shown to bind to CdSe nanocrystalsvia the hydroxyl groups and acetone has been demon-strated to be a stable ligand under BH4

− and HCl conditions[25].

Polyols and ene-diols, bearing multiple alcohol groups,are capable of binding to NPs composed of transition metaloxides [95]. Dopamine and ascorbic acid [96,97] co-ordinateFe atoms in iron oxide NPs in a favourable octahedral manner

(Fig. 2). Meanwhile in the polyol process for reduction ofionic NP precursors in organic solution hexadecane-1,2-diolis also used as a multico-ordinate ligand [35].

Polyols can be used as a selective binding agent in blockpolymer ligands; the double hydrophilic block copolymer,poly[2-(methacryloyloxy)ethyl phosphorylcholine]-block-(glycerol monomethacrylate) binds to Fe3O4 NPs via theglycerol monomethacrylate block [75]. Meanwhile thezwitterionic phosphorylcholine groups do not compete withthe glycerol for NP binding, but instead provide watersolubility through the charged groups.

More common cases of neutral oxygen donor ligands arePEG and carbohydrates such as glucose, sucrose, and starch.However, as PEG itself is simply a polyether, it must gener-ally be modified to bear a more reactive terminal functionalgroup to serve as an anchor to the NP.

Glucose and sucrose have also been used in the reductionand post-synthesis stabilisation of aqueous salts of Ag andAu [98]. Starch may likewise be used for the stabilisation ofnoble metal and iron oxide NPs [29].Anionic ligands. This class of ligands is composed predom-inantly of carboxylic acids and their derivatives. Two of themost well-known of these ligands are the hydrophilic citrateanion used to cap Au NPs and the hydrophobic oleic acid(OA), used in the synthesis of Fe-containing NPs in organicsolvents. Oleic acid is a particularly ubiquitous carboxylicacid as it also finds use in binding and stabilising almostall core NPs. Other examples of carboxylic acid stabilis-ers include poly(acrylic acid), poly(methacrylic acid) andderivatives [3,80,99].

Hydroxamic acids have a high affinity for many metaloxide surfaces and can be used to introduce dendrons tothe surface of NPs, providing a high degree of protectionagainst acid etching [100].

Nitrogen-based ligandsNeutral ligands. As well as acting as an organic-aqueousPT agent, zwitterionic DMAP can be used to cap Au NPs [34].Unlike most nitrogen-based PT agents (see below) DMAP isproposed to bind to the NPs via the nitrogen atom bear-ing the negative charge in the zwitterionic form of themolecule.

The bidentate 5-methyl-6-carboxy-2,2′-bipyridine iscapable of binding to La0.95Eu0.05F3 NPs, replacing the2-aminoethane phosphate ligands used in the synthesisprocedure [101]. The ligand assumes an energeticallyfavourable tridentate binding mode, with both nitrogenatoms and the carboxylate group binding to the NP surfacethus explaining why, despite the presence of polar groups inthe ligand, such NPs are insoluble in water.Cationic ligands. Tetraalkylammonium salts have a varietyof applications in the synthesis and stabilisation of NPs. Forexample, TOAB is used as a PT agent and ionic stabiliser dur-ing the two-phase synthesis of Au NPs, which are colloidallystable for up to two weeks without further functionalisation[33]. Similarly, tetramethylammonium hydroxide providesionic stabilisation of aqueous Fe3O4 NPs prepared by alkalicoprecipitation, protecting them from aggregation for upto a week. However, these ionically stabilised NPs are verysusceptible to salting out in the presence of electrolyte con-centrations well below physiological conditions.



Alkylammonium salts are also used as templates in theformation of mesoporous silica shells on NPs. The most com-mon reagent for this purpose is cetyltrimethylammoniumbromide (CTAB), which may additionally be used as a PTagent [86,102].

Phosphorous-based ligandsOne of the most important phosphorous-based ligands isTOPO. It is used primarily during the synthesis of semicon-ductor QDs, either as the solvent or to provide colloidalstability in the chosen organic solvent. PEG-based ligandsbearing phosphine oxide groups provide stabilisation andsolubilisation for NPs composed of transition metal oxides,semiconductors and noble metals [91,95].

Organophosphates and phosphonates are suitable forbinding to a range of surfaces similar to those accessibleby phosphine oxide ligands [1,64,103,104]. However, due tothe additional bulkiness of the phosphate group compared tothe carboxylate group, the reported coverage of phosphatesis around an order of magnitude lower than for carboxylicacids [1].

The orthophosphate anion, PO43−, has a relatively low

affinity for NP surfaces at pH values greater than 5, but canadsorb well onto iron (hydr)oxides at low pH [47]. This isparticularly important as almost all ligand-stabilised NPs forwhich stability data are available were studied in solutionsof phosphate buffered saline (PBS).

Phosphines (e.g. triphenyl phosphine) may serve as labileligands for Au NPs; providing steric stabilisation yet beingexchanged readily for ligands with higher affinity for themetal surface [24,105,106].

Sulfur-based ligandsNeutral ligands. Alkane thiols make good soft Lewis baseswhich makes them complementary to the soft acid prop-erties of NPs composed of Au, Ag, Pt, Pd and their alloys[25,107,108]. With Pd NPs care must be taken with thecorresponding ligand shell, as they are less stable thanAu NPs, and they will decompose upon exposure to air[109].

There is evidence to suggest that even with the use ofthiol terminated ligands, disulfide-like bonds form on theNP surface [109]. However, NPs with monolayers preparedfrom either species are indistinguishable [82].

It is also reported that disulfide ligands provide Au NPswith enhanced colloidal stability compared to monothiols[110,111], suggesting that the choice of thiol or disulfidecould be used to tune the stability of the ligand shellwithout significantly affecting the packing of the ligandmolecules.

It has been shown that mono-, dithiol and amine termi-nated peptides provide superior stability to peptides with nothiol groups [3,48]. Similarly the incorporation of a thioethergroup to the stabilising agent poly(methacrylic acid) (PMAA)allowed cobalt NPs to be stored for up to eight weeks asopposed to only 11 days with pure PMAA. This indicates theimportance of thiol groups in stabilising cobalt NPs.

In view of the fact that QDs containing S, Se and Te areunstable in the presence of a monodentate thiol, polythiolligands have been successfully employed as capping agents[112,113].

It has been demonstrated that NPs can be coated witha self-assembled and highly ordered monolayer composedof a binary mixture of ligands with a hydrophobic content ashigh as 66% [114]. Boal and Rotello [115] exploited this ligandself-organisation to create a self-assembled two-componentrecognition site for binding flavin via hydrogen bonding and�-stacking.

As well as introducing additional chemical functionality,thiol-modified biofunctional species may be added directlyto the NP surface in this manner, for example, DNA bearing a3′ or 5′ thiol modification can be conjugated to Au NPs [105].

Other sulfur-containing functional groups such as thiocar-bamates and xanthates can be used as ligands for a varietyof transition metal-based NPs, but they have lower affinityfor the NP surface than thiols [82,116]. On the other hand,xanthates can provide high protection for Au NPs againstetching by cyanide.Anionic ligands. Sulfonates are capable of binding to ironoxide [104] but their binding affinity to Au surfaces islower than that of thiols [114]. Micelles formed from theanionic surfactant bis(2-ethylhexyl) sodium sulfosuccinate(AOT) can be used as a surfactant in the synthesis of AgNPs [117,118]. AOT is a labile ligand, which could be ligandexchanged to introduce chemo and biofunctional species.

Other factors affecting ligand shell stabilityThe stability of ligand shells is governed by many of the samefactors that stabilise self-assembled monolayers. Stability isdominated by the strength of the ligand-surface bond, butthe ability of the ligand tails to pack in an ordered fashionis also significant. The structure and hence the stability of aligand shell can be altered by the position of a single methylgroup [119], the presence or absence of a single unsaturatedbond [120] or other packing factors such as chain length[121] and �—� stacking [108]. The concentration of free lig-ands available to form a monolayer also influences the ligandorientation and hence degree of stabilisation afforded [122].Light induced reactions at the nanocrystals/ligand interfacecan also often lead to desorption of the ligands [123].

Ligand addition

Ligand addition involves a modification of the externalsurface of the NP-ligand shell without removal of any pre-existing ligands. The four approaches to ligand addition areoutlined as follows: (1) addition of ligands to NPs initiallyprepared with no capping agent; (2) indirect ligand addition;growth of a layer of inorganic material such as amorphousor mesoporous SiO2, Au, iron oxide, carbon, onto the NP sur-face with subsequent adsorption of a ligand species directlyonto this surface by ionic or other non-specific interactions;(3) exploitation of the ‘‘hydrophobic attraction force’’ tointercalate hydrophobic species into the hydrocarbon shellof NPs capped by ligands such as OA and (4) formation of acovalent bond between the existing ligand and the incomingligand.

Core—shell structuresAs expected, ligand-NP affinity is highly dependent on theNP surface and the ligand head group. In many cases theNP core is chosen for its desirable physical properties such



Figure 3 Simplified scheme of silanisation reaction of APTES on NP surface, MPTES differs only by having —SH end groups asopposed to NH2.

as electronic, magnetic or optical behaviour, but presents asurface incompatible with the chemical functionality of thedesired ligand. In order to overcome this, the surface canbe coated with a thin shell of material for which the ligandhas a high affinity [124].

While this strategy is suitable for creating NPs with com-posite attributes, it must be noted that physical propertiesof the core such as saturation magnetisation, MS, can dete-riorate with increasing shell thickness. As little as four extramethylene groups in a ligand tail can have a large impact onthe measured magnetic properties [125]. Meanwhile, ZnSa shell can also improve the properties of semiconductingmaterials such as CdSe [126].

The NP shell can provide protection for the core, addedmodes of functionality or introduce different chemicalgroups for further functionalisation, in the following sec-tions we have outlined a variety of shells that have beeninvestigated.Amorphous silica. Two main silicon-based ligands are fre-quently used to change the surface chemistry of NPs,silica (SiO2) and trialkoxysilylpropane bearing a functionalgroup on the 3 position of the propyl chain. Commonexamples of the latter are (3-aminopropyl)triethoxysilane(APTES) (Fig. 3), the trimethoxy analogue APTMS and (3-

mercaptopropyl) triethoxysilane (MPTES). These silanes canbe used to introduce surface functionality for further chemoand biofunctionalisation.

Using a modified version of the Stöber process, a layer ofamorphous silica may be grown on the surface of NPs [20].Modern methods involve the base-catalysed hydrolysis of atetraalkyl orthosilicate, commonly tetraethyl orthosilicate(TEOS) in a 4:1 mix of ethanol:water; the thickness of thesilica shell can be controlled by altering this ratio [27]. Thegrowth of amorphous silica depends very strongly on the pHof the reaction mixture. At low pH, NPs become encasedin a silica matrix, at mildly alkaline pH (7.5) core—shellstructures are formed with individual NP cores coated bya thin layer of silica. At high pH, a mixture is formedcontaining the original NPs and silica NPs with no coatingobserved. An alternative process proposed by Graf et al.[69] involves coating NPs in PVP. The PVP-coated NPs canthen be adsorbed onto the surface of aminated silica NPsand a further layer of SiO2 is then deposited by hydrolysis ofTEOS.

By using a silanisation agent such as APTES instead ofTEOS, a very thin layer of SiO2 may be deposited on the sur-face of the NPs. This layer of SiO2 is terminated with thefunctionalised propylene groups, effectively allowing the

Figure 4 Use of CTAB in the conversion of hydrophobic NPs into mesoporous ‘cargo transfer agents’.



introduction of any surface functional group via the sameligand-core chemistry. This functionality can be introducedeither before or after the silanisation is carried out [58].Mesoporous silica. Alternatively, mesoporous SiO2 may begrown on the surface of NPs, generally after the depositionof a thin layer of amorphous SiO2 as described above. Thepresence of a templating agent such as cetyltrimethylammo-nium bromide (CTAB) leads to the formation of mesoporesaround 2—4 nm (Fig. 4). This allows for the loading of drugs,e.g. doxorubicin, ibuprofen, and other molecular species[58,102]. Not only can such pores be used to deliver cargo,but they can also be used to scavenge biological species suchas microcystins [127].

Shells of mSiO2 can be deposited on NP surfacescomposed of metal oxides [58], semiconductors [102], amor-phous SiO2 [127], and Au [128]. The emission spectrum ofthe QDs encapsulated with mSiO2 by Kim et al. [102] is redshifted relative to the free QDs, suggesting that the QDs areheld in close proximity within the silica shell. It should benoted that such encapsulation using amorphous SiO2 is pHdependant [128].Gold and other noble metals. Procedures for the prepara-tion of Au NP structures include seed mediated growth andsacrificial reduction. In seed mediated growth, Au ions arereduced in the presence of the NP cores requiring alteration[86,129,130]. However, where TEM evidence is presentednucleated nano-structures are formed. This occurs becausethe surface of the core NP is suited to nucleation of the noblemetal (i.e. Au and Ag). Due to the high interfacial energy,the noble metal grows outwards from the ‘‘core’’ forminga twinned structure rather than forming an encapsulatinglayer [86,131].

Sacrificial reduction, as the name suggests, involves thesacrifice of the outer layer of the NP core, which acts as areducing agent to Au3+ ions in solution. Oxidised core mate-rial is lost to the solution which leads to a decrease in sizeof the magnetic core and lower coercivity and saturationmagnetisation values. However, thanks to loss of some ofthe initial core material the resulting NP size is roughly thesame after coating as before.

Amine terminated SiO2 NPs can be used as frameworks.Fe3O4 NPs were grown on amine terminated SiO2 NPs fol-lowed by the deposition of Au seeds (1—3 nm) and a 15 nmAu shell [86,132]. Liz-Marzan and his team have also con-tributed a wealth of knowledge concerning the coating ofNPs with SiO2, a review of some synthetic methods for coat-ing Au and Fe3O4 and the topological effects on materialfunction has been published and this work has since beenadvanced [132].Semiconductors. Transfer of QD semiconductors to aque-ous phase has been covered elsewhere [133,134]. By coatinga narrow band-gap semiconductor such as CdSe with a largeband-gap layer such as ZnS, electron/hole pairs generatedby absorption of photons are confined to the core by thepotential difference between the electronic bands of thecore and shell thus preventing photon emission. By deposit-ing a shell of ZnS on CdSe QDs, the quantum yield canbe increased from about 10% to 50—60% [126,135,136]. Byadopting a large initial Se:Cd ratio of the precursors, it isalso possible to gain QYs of up to 80% [137]. Coating �-Fe2O3

with a CdSe/ZnS shell has also been performed and offersthe advantage of dual functionality [138]. The ZnS also offers

the advantage of a physiologically stable barrier which pre-vents hydrolysis of the core and dissolution of poisonous Cdinto the bloodstream.Iron oxide and other magnetic materials. Due to theincreased ease of biofunctionalisation of iron oxide, itis commonly used to coat other magnetic materials suchas FePt [139,140], Co and SmCo5.2 [96]. These shells aredeposited by methods similar to the preparation of Fe3O4

NPs by the thermal decomposition of Fe(CO)5 followed byoxidation, or reduction of iron salts such as Fe(acac)2. Theadvantage of this technique is that non-biocompatible mate-rials with magnetic properties superior to magnetite maybe encased in iron oxide while retaining enhanced magneticbehaviour.Carbon. Bulk carbon is a bioinert material which makes ita suitable material for NP shells. However, there are somesuggestions that nanoparticulate amorphous carbon may dis-play higher cytotoxicity than the bulk [141]. There are alsowell-published concerns about the health risks of carbonnanotubes and fullerenes, discussion of which is beyond thescope of this review. Carbon-coating of NPs can be carriedout by addition of a carbon-bearing ligand followed by heat-ing until the ligand is carbonised [55]. This technique hasbeen proposed to be suitable for drug delivery, but no evi-dence has yet been published to support this idea.

Hydrophobic interactionsThe phospholipid membrane bilayer structure in living sys-tems is well known and there is evidence for similarstructures forming during the capping of NPs with ligandsbearing long hydrocarbon chains [103]. In these situationsa complex system of forces and interactions attributed tothe ‘‘hydrophobic effect’’ causes the hydrocarbon chains tointerlace, forming entropically favoured structures such asmicelles and bilayers [45].

These interactions can be exploited to attach otherspecies bearing unreactive hydrocarbon chains without priormodification of the chain. Poly(maleic anhydride-alt-1-tetradecene) bears hydrophobic C14 chains on every otherrepeat unit, allowing it to form an external shell aroundNPs capped with hydrophobic surfactants. The polymer canthen be cross-linked with a hydrophilic agent allowing theNP to be further modified under aqueous conditions [142].This approach is applicable to all core types as the onlyrequirement is the hydrophobicity of the ligand tail group[70,142].

Phospholipids, e.g. phosphatidylcholine and phos-phatidylethanolamine are zwitterionic, bearing bothphosphate and ammonium groups. Such ligands can formmicelles with a 5 nm hydrophobic interior capable ofencapsulating TOPO-capped NPs leaving the hydrophilichead group exposed for further functionalisation after PTto water [60,143,144]. This class of nanostructure is knownas solid lipid nanoparticles (SLNs).

An alternative to SLNs is to encapsulate multiple NPsin the hydrophobic interior of larger liposomes [145]. Thisapproach has the advantage that liposomes are biocom-patible and may fuse with the cell wall, allowing forrelatively facile transfection. Additionally, the liposome maybe loaded with a cargo species without having to bind thedrug to the NPs directly (Fig. 5). This means that the cargo



Figure 5 Use of liposomes as magnetic drug delivery moieties, D represents a chosen drug.

can be magnetically targeted, but requires no chemicalmodification.

Effects of the ligand shell on the physical andbiomedical properties

ToxicityLigands are essential to the reduction of cytotoxicity ofmany NPs, as uncoated NPs are generally cytotoxic them-selves [146—148]. It is likely that significant quantities ofNPs will be taken up by phagocytes and macrophages, sothey must be protected from the harsh chemical environ-ments present through the employment of a chemically inertshell [149]. However, ligands do affect the properties of NPsand these changes should be noted. A study investigatedthe genotoxicity of amine terminated FePt NPs in the Amestest and in vitro chromosomal aberration test, but furtherstudies are required to establish whether a positive aberra-tion result is irrelevant [150]. Details of interaction of NPswith the immune system have been published in a recentmini-review [151]. The Stellacci group have also presenteda critical review of current understanding of how syntheticand natural chemical moieties on NPs interact in the bodyand the challenges of systematic studies [152]. For informa-tion detailing in vitro studies investigating the cytotoxicityof metal and semiconductor nanoparticles please see thereview by Drezek et al. [153].

Magnetic propertiesAs ligands bind to NPs by donation of electrons it is inevitablethat there will be some effect on the electronic state ofthe NP, at least near the surface. Tanaka and Maenosono[154] studied a variety of thiols, amines and carboxylic acidsand report that the saturation magnetisation of the NPsis significantly lower than that of the bulk phase and theuncapped particles, with carboxylic acids showing the great-est decrease for the FePt alloy NPs studied.

However, there is some confusion over the extent of thiseffect due to variations in reporting magnetic data for NPs.When the ligand layer is substantial, there will clearly be alarge difference between magnetisation values reported perunit mass of NPs [27,38,64,75,96,155] and that of core mag-netic material. There is also evidence that non-magneticshells can alter the physical and chemical nature (i.e. oxi-dation states) of atoms in the core of the NPs [129,156].

Optical propertiesThe addition of a ligand layer to semiconductor QDs canbe used to passivate the surface, thereby enhancing thequantum yield [135]. Surface passivation can be carriedout using species such as hexadecylamine [126], mercap-tosuccinic acid [21] or PVP [157]. Conversely, conjugationof Au NPs to QDs leads to quenching of luminescence byaround 70%. Cleavage of the QD-Au tether and the subse-quent increase in luminescence could be used as a probe forreal-time monitoring of biochemical events such as proteol-ysis [93].

Alternatively, the ligand shell may act as an antenna,harvesting light and delivering it to the NP core. This sen-sitization can lead to a large increase in the absorbance orluminescence spectra of the NPs [101,108,158].

Biofunctionalisation

Common biofunctional species and their uses

Nucleic acidsThe specific nature of DNA complementary binding isexploited in colorimetric assays for gene detection [159].This strong, specific, non-covalent binding can also beemployed to attach DNA-tagged NPs [60,105,160] to the sur-face of other NPs. However, it is worth noting that DNA isalso capable of non-specific binding interactions with NPs,leading to less selective complementary binding interactions[161]. Non-specific binding of QDs to DNA has been shown tobe entropically driven, this behaviour is frequently observedbetween proteins and DNA; such QDs have been labelled‘inorganic proteins’ [162]. Functionalisation of QDs withhydroxyl groups reduces solubility issues encountered whenattempting to detect chromosome abnormalities or muta-tions when using fluorescence in situ hybridisation (FISH)procedures [163].

ProteinsLike nucleic acids, proteins are known for their specificbinding interactions. However, nucleic acids are limitedto interactions with other nucleic acids whereas proteinscan interact with a wide range of substrates and syntheticanalogues [146,164,165]. Another advantage of proteinsis their enzymatic activity, which may be harnessed incatalytic applications [166]. In bionanotechnology, specificfunctions of proteins such as antibody—antigen detectionand receptor-substrate recognition such as the biotin—avidin



interaction are very useful. They can either be used toimmobilise species on the NP surface (i.e. as an intermedi-ate in the functionalisation process) [167] or constitute thedesired functionality of the NP itself (e.g. for immunoas-says or targeted NP delivery) [87,146,165]. Proteins can beincorporated into NPs during the coprecipitation step, in areported one-step synthesis and biofunctionalisation [168];however, there is little evidence that such treatment leadsto retention of the protein function. More sophisticated bio-conjugation methods are desirable to make sure that theprotein is bound to the NP surface in such an orientationthat the biological activity is retained. Such methods arediscussed later in this review. An added bonus of proteinssuch as albumin [56] and transferrin [83] is that they mayalso be used in order to reduce cytotoxicity and facilitatecellular uptake of NPs. Another consideration when bindingproteins to NPs is that these molecules are very large (gen-erally tens to hundreds of kDa, or a few nm across), so thiscan add a relatively thick ligand shell [87,169]. In order tokeep the hydrodynamic radius small enough for in vivo appli-cation it is desirable to conjugate only the active fragmentof the protein to the NPs [170,171].

PeptidesNatural and synthetic peptide ligands both have poten-tial for the stabilisation and biofunctionalisation of NPs.Such biofunctional peptide sequences include ‘‘membranetranslocation signals’’ like the HIV-Tat peptide sequence[172,173], which is capable of transporting nanoscalematerials (proteins and NPs) across cellular membranes.Additional peptide sequences such as ‘‘nuclear locating sig-nals’’ could be used for further intracellular targeting [84].While peptides such as CALNN may provide stabilisation inaqueous solutions [48], there is evidence of CALNN-cappedNPs aggregating when endocytosed [84]. This could be over-come by combining peptides with other steric stabilisationagents such as PEG.

PhospholipidsPhospholipids are inherently suited to encapsulating bothhydrophobic and hydrophilic payloads, allowing them toserve as the outer envelope of nanocomposite structureswith potential for drug delivery and hyperthermia appli-cations [174]. Encapsulation of silica NPs has been shownto reduce non-specific interactions thanks to the bio-compatibility of the phospholipid head group. As well asbiocompatibility, the head group offers the possibility forbiofunctionalisation, e.g. biotinylation, allowing for furtherbioconjugation steps exploiting biotin—avidin interactions[175].

CarbohydratesCarbohydrates such as dextran have long been used in bio-compatible iron oxide NPs for MRI enhancement thanksto their low cytotoxicity. There is some evidence to sug-gest that dextran-coated NPs may still induce cytotoxiceffects — although the severity is reduced compared to theuncoated NPs [56]. However, dextran-coated iron oxide isgenerally claimed to be especially non-toxic as the wholesystem degrades in vivo, being safely metabolised and elim-inated [176]. Another advantage of dextran is that it may

be aminated using ammonia, allowing facile conjugationwith complementary chemical groups [172]. Chitosan canbe used as a biocompatible coating for a range of NP corematerials and, like aminated dextran, it presents aminefunctionality for subsequent attachment of biofunctionalligands [57,177]. Starch vermicelli has been used as a tem-plating agent in the formation of Au and Ag NPs which canbe heated to obtain potentially biomedically useful carbon-coated nanostructures [55,178].

Including starch in the alkali coprecipitation processleads to the formation of small NPs aggregated into clustersof 200—300 nm in size, such clusters have potential in thepreparation of iron oxide-carbon nanocomposite materials[55].

Smaller carbohydrates such as lactose, glucose, mannose[179—181] can be thiolated for attachment to Au NPs by lig-and exchange. The particles prepared this way may be usedas sensitive colorimetric probes for a variety of metal ions.Mannose and lactose have also been used for the reductionof Au and Ag salts and stabilisation of the resultant NPs [98].

Closely related to carbohydrates, oligosaccharide aldonicacid shells can be used to alter cellular uptake of NPs,with maltotrionic acid leading to reduced cellular uptakewhereas lactobionic acid increases uptake [182]. Again,these NPs exhibit low cytotoxicity at NP concentrations of200 �g mL−1.

Coupling strategies for biofunctionalisation

Carbodiimide couplingWhile it is possible to form amide bonds directly between aterminal carboxylic acid group on the surface of a NP and afree amine on a biological species like proteins [83], reactionefficiency can be enhanced through the use of additives.

Carbodiimide coupling is used to covalently link car-boxylic acids to amines via formation of a ‘‘zero length’’amide bond [73]. The key advantage of this procedureis that it involves no lengthy linker species, allowing thehydrodynamic radius of the NP to be minimised. The mostcommon carbodiimide coupling strategy used 1-ethyl-3-(dimethylaminopropyl) carbodiimide hydrochloride (EDC orEDAC) (Fig. 6) as the coupling agent.

This strategy has been applied to coupling of enzymesto NPs, with retention of up to 50—80% of enzymaticactivity, depending on the enzyme [169]. The efficiencyof the coupling reaction can be increased by stabilisingthe O-acylisourea intermediate by formation of the suc-cinimide ester. This is achieved by addition of NHS orsulfo-NHS. Rather usefully bis-N-hydroxysuccinimide can beused without a carbodiimide activation agent allowing theconjugation of two amines thanks to the two NHS estergroups present [165].

EDC-activated species may be coupled to existing ligands[183] or, if the synthesised NP bears hydroxyl groups on thesurface, the activated species may be directly coupled tothe NP surface via a net dehydration reaction, leading to anester linkage as opposed to an amide [146,167,184].

Maleimide couplingA maleimide may be used to conjugate primary amines tothiols as illustrated in Fig. 7 [185]. The most commonly used



Figure 6 Schematic of carbodiimide coupling of an acid to an amine using EDC as the coupling agent. An N-hydroxy succinimide(NHS) ester intermediate may be formed to increase reaction efficiency.

Figure 7 Maleimide coupling of an amine and a thiol using sulfo-SMCC as the linker.

maleimide-derived coupling reagent is sulfosuccinimidyl-4-(maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC).

Maleimide coupling has been used to conjugatebiomolecules such as DNA [60], herceptin [87] and proteins[164,186] to NPs. Lee et al. [187] used maleimide couplingto link iron oxide and dye-doped silica NPs, creating MRI-and fluorescence-active imaging agents.

Click chemistryAnother common route for bioconjugation, the Cu(I)-catalysed alkyne-azide cycloaddition reaction also known as‘‘CuAAC’’ or ‘‘click chemistry’’ involves the coupling of analkyne to an azide giving a 1,2,3-triazole ring, which servesas a strong covalent bond between the NP and the biofunc-tional moiety (Fig. 8). This process has been demonstratedto be highly versatile, suitable for conjugation of a varietyof species including small molecules.

This procedure provides versatility through the fact thateither the alkyne or the azide could be expressed on the bio-functional moiety, with the complementary functional groupexpressed on the NP surface. Combined with the variety of

Figure 8 Schematic of Cu-mediated alkyne-azide cycload-dition (‘‘click reaction’’). The reaction proceeds via a Cu(I)intermediate (CuSO4) [1].

ligand head groups available for NP-ligand bond formation,this procedure has a lot of potential as a coupling approachfor bioconjugation [1]. Furthermore, the one-step click pro-cess has been shown to give the possibility of introducingmultiple functionalities onto the NP [188]. However, theprocedure does have the disadvantage that preparation ofthe azide or alkyne-functionalised bioactive species is oftenlengthy and low yielding [189]. So, while the final cycload-dition step itself proceeds rapidly and with high yield, theoverall functionalisation procedure can end up being longand inefficient.

Disulfide bridgesDisulfide bridges have been used for the reversible chemicalcoupling of NPs. Tian et al. [190] showed that the oxidationand reduction of disulfide bridges between silica NPs andFe3O4 NPs was facilitated by glutathione disulfide (oxidativebond formation) and dithiothreitol (DTT) (reductive bondcleavage). While current work appears to be focussed onthe formation of hybrid nanostructures, this approach couldalso be adapted for drug delivery because cleaving agentssuch as glutathione (GSH) [191], are present at appreciableconcentrations in vivo.

Histidine tagged proteinsThe addition of six terminal histidine residues (‘‘6 His tag’’)on a protein allows the protein to act as a chelating agent,coordinating to a metal cation held to the NP surface bynitriloacetic acid groups. This type of binding interactionis utilised in the magnetic assisted purification of proteinsextracted directly from cell lysate [96,107] and specificenzyme binding to the surface of Au NPs giving almost



complete retention of enzymatic activity [90]. This bindinginteraction is capable of detecting 6 His-tagged proteins atconcentrations as low as 0.5 pM without non-specific bindingto undesired proteins occurring [107].

Ionic couplingCharged NPs may be coupled either with oppositely chargedbiological and polymeric species [192], or indeed to differ-ent oppositely charged NPs [193]. Some obvious examplesof biological application are the coupling of negativelycharged DNA [30,194] or liposomes to positively charged NPs[195,196]. With careful tuning of the pH it is possible tocouple a variety of proteins, which can be either cationic,anionic or neutral, to the surface of oppositely charged NPs.Immunoglobulin G (IgG), a cationic species, can be immo-bilised onto the surface of citrate stabilised Au NPs at pH 6[167].

Radionuclides [172] and imaging agents such as Gd [197]may also take advantage of ionic coupling to chelatingagents allowing another means of NP detection or makingway for a therapeutic guided delivery of radioactive com-pounds.

Specific bio-recognition interactionsIn order to retain the specific recognition ability of antibod-ies, it is desirable to bind the antibody in a fixed positionrelative to the NP so that the antigen binding site in the Fabfragment remains exposed. If the antibody has multiple anti-gen binding sites in the Fab fragment it is sometimes possibleto bind the antibody to the NP via an active site irrelevantto the biomedical application. This is demonstrated by Hoet al. [167], in which protein G is bound to the NP surface,immobilising IgG via interactions with the Fc region of theantibody but leaving the Fab region of the protein free forspecific in vitro recognition of Staphylococcus bacteria.

Complementary binding of base pairs is exhibited in dou-ble stranded DNA and it is desirable to emulate this inbioconjugation. For example, nucleotide specific interac-tions were used for reversible assembly of DNA conjugatedNPs by Mirkin et al. in 1996 [198]. Through attachment of sin-gle stranded DNA to NPs, self-organisation was also achieveddimers and trimers of NPs [105]. A protocol for the conju-gation of specific DNA aptamers onto Au NPs was reportedto give an optical response upon the binding of differentanalytes [199].

Avidin and analogues such as streptavidin and neutra-vidin are capable of forming up to four interactions withbiotin, making it an ideal cross-linker. Such functionality canbe exploited through the conjugation of biotinylated PEGor phospholipids to NP surfaces [68,175,200,201] which canthen interact with avidin or avidin-functionalised species.Synthesis of biotinylated NPs specific to free avidin couldmake good avidin sensors [68] or the incorporation of avidinspecies into the NP allows the possibility of using the site foraddition of further biotinylated species to form a ‘‘B—A—B’’linkage. While this is a strong and specific non-covalentinteraction suitable for the formation of 3-D nanostructures[200,202], a significant setback is that a B—A or B—A—B link-age is long, adding significantly to the hydrodynamic radiusof NPs and reducing their suitability for some in vivo appli-cations.

Biomedical applications

The small nature of NPs allows them to cross cellularmembranes and avoid detection by the reticuloendothelialsystem and their high surface area to volume ratio can allowincreased loading of therapeutics; such properties makesNPs desirable for diagnostic and therapeutic applicationswhich are briefly detailed, as follows [133,203,204].

NPs are employed for imaging in a variety of ways,both for medical purposes and further understanding ofbiochemical processes in vitro and in vivo [205]. Generalreviews covering the applications of a variety of materialsfor biomedical imaging including Au, QDs, and MNPs havealready been published elsewhere [148,206].

After Faraday studied finely divided Au in 1857, littleattention was paid to its photophysical properties untilTurkevich et al. published a paper detailing the nucle-ation and growth processes of colloidal Au in 1951. Thisresulted in a wide expansion of research into Au NPs tofurther our understanding and establish possible biomed-ical applications. The plasmon absorption and scatteringproperties of Au NPs make them favourable materials forimaging and sensing [159,207—211]. The author is directedto a review published by Boisselier and Astruc for exten-sive coverage of Au NPs including use of surface enhancedRaman scattering (SERS) imaging which has been used fortargeting and imaging of cells and cancer markers [66].Au NPs also find application in therapy; light has beendemonstrated as a good orthogonal stimulus to effect drugrelease from gold NPs acting as both cage and carrier[212]. Au NPs with engineered surfaces have been shownto aggregate in low pH environment resulting in a NIRshifted absorption band; this shift has been exploited toyield a new proof of concept for photothermal cancer ther-apy [213]. Recently, site selective assembly of MNPs onto Au rods yielded NPs with tunable optical and magneticproperties, these probes were shown to exhibit simultane-ous detection, separation, and thermal ablation of multiplepathogens [214]. Through the attachment of an antibodyand platin complex via a thiol based linker onto the goldpart of dumbbell shaped Au-Fe3O4 nanoparticles, targetspecificity and strong therapeutic effects have been demon-strated in the treatment of Her2-positive breast cancer cells[215].

The semiconducting nature of QDs gives them excel-lent, tuneable, photophysical properties [216]. Labellingof cellular proteins, cell tracking, pathogen and toxindetection, in vivo animal imaging and fluorescence reso-nance energy transfer (FRET), are all techniques that havebeen developed to take advantage of these properties;please see previously published reviews and articles for fur-ther information regarding biological applications of QDs[7,112,134,217—224]. Diffusion dynamics of glycine recep-tors have been revealed by single quantum dot tracking, thusdemonstrating the power of QDs in furthering our under-standing of in vivo molecular dynamics [225]. Photodynamictherapy (PDT) uses the combination of a photosensitizingdrug and light in the presence of oxygen to cause selectivedamage to target tissue. There is growing interest in thepotential for QDs to act as FRET donors in conjunction withconventional photosensitizing agents for PDT. QDs possess



superior properties to organic photosensitizers including abroad absorption spectra; these attributes are all describedelsewhere [226].

Magnetic NPs have long been used as contrast agents formagnetic imaging; the reader is directed to recent reviewswhich cover progress towards improved contrast agentsderived from Fe, Co and Pt for MRI and MPI applications[5,53,66]. Research has been growing in cellular MRI (CMRI);this type of cell tracking shows potential in the evaluationof novel drug therapies [206]. Apoptosis of tumour cells wasreportedly detected with MRI using a contrast agent in 2001[227]. Resolutions of up to 1 mm have been achieved bytaking direct signals from magnetic NPs; magnetic particleimaging (MPI) technology also has the advantage that it doesnot require bulky full body scanners [5].

Drug delivery systems provide an important tool forincreasing the efficacy of pharmaceuticals through improvedpharmacokinetics and biodistribution. MNPs have beeninvestigated as platforms for transport of drugs and genes;delivery can be performed via passive, active or directmeans [228]. Research into the functionalisation of MNPsthrough different coatings and structures with emphasison the active targeting approach continues to yield results[53,229].

The low pH conditions inside breast cancer cells canreportedly cause acid etching of herceptin conjugatedporous Fe3O4 NPs to further open the pores to give a regu-lated release of cis platin into the cell; this result indicates apossible delivery formulation for target specific therapeuticapplications [230]. Magnetic separation technology has beenimplemented into a continuous flow microfluidic device toseparate individual cells and can be observed visually usinga low power microscope [231].

Magnetic actuation for in vitro non-viral transfectionand tissue engineering, in vivo drug and gene delivery,and recent clinical results for magnetic hyperthermia treat-ments by direct injection of amino silane coated iron oxideNPs are presented by Pankhurst et al. [5]. Efforts are con-tinuing in the ultimate goal of functionalising MNPs fortarget specific hyperthermia treatments [3,4,232]. Analy-sis has been done on single intracellular NPs to establisha mechanism of action for intracellular heating; this workindicated the formation of short lived ‘nanobubbles’ whichhave potentially explosive behaviour in cells and could beused for future cancer therapy [233]. By taking a synergisticapproach, incorporation of cytotoxic carboplatin into Fe@C-loaded chitosan magnetic NPs followed by thermal inductionhas shown efficient destruction of tumour cells [234].

Conclusion

In order to successfully prepare and biofunctionalisenanoparticles for a given biomedical application, a widerange of physical, chemical, biological and physiologi-cal factors and conditions must be taken into account.However, by tuning the nature of the core, shell and lig-ands, these factors can be taken advantage of to providethe desired, biocompatibility and biofunctionality, makinginorganic nanocrystals suitable for a very wide range ofapplications in diagnostics and therapy for numerous medi-cal conditions.

Acknowledgements

Ian Robinson, Le Trong Lu, Daniel Dawson, Le Duc Tung andCristina Blanco-Andujar are acknowledged for their help-ful discussions and assistance. Nguyen T.K. Thanh thanksthe Royal Society for her Royal Society University ResearchFellowship. Luke Green is sponsored by a UCL-RI PhD stu-dentship.

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Dr Nguyen T.K. Thanh, FRSC, CChem, CSci,MRI, received the award for top academicachievement in Chemistry and was selected tostudy at the University of Amsterdam for herMSc in 1992. In 1994—1998, she read Biochem-istry in London for PhD. In 1999, she undertookpostdoctoral work in medicinal chemistry atAston University, UK. In 2001, she moved tothe United States to take advantage of pio-neering work in nanotechnology. In 2003, shejoined the Liverpool Centre for Nanoscale

Science. In 2005, she was awarded a prestigious Royal Society Uni-versity Research Fellowship and University of Liverpool Lectureship.In January 2009, she was appointed a UCL-RI Readership in Nan-otechnology and based at the Davy Faraday Research Laboratory,The Royal Institution of Great Britain and Department of Physics andAstronomy, University College London. She leads a research teamfocused on the design, synthesis and study of the physical proper-ties of nanomaterials as well as their applications in biomolecularand biomedical research.

Mr Luke A.W. Green, MChem, AMRSC, MRI,obtained his MChem with a year in Europedegree from the University of York in 2008.After a short spell working at a hospital bio-chemistry department, he is now studying fora PhD under the guidance of Dr Nguyen T.K.Thanh at UCL and is based at the Royal Insti-tution of Great Britain. His interests are inthe synthesis of magnetic nanoparticles forbiomedical applications.

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